SEQanswers

Go Back   SEQanswers > General



Similar Threads
Thread Thread Starter Forum Replies Last Post
QuantSeq FWD HT has been launched! lexogen Vendor Forum 0 02-22-2017 07:32 AM
Total RNAseq multiple mapping issue wri_ichp RNA Sequencing 2 02-11-2016 11:16 PM
Did low input library and traditional library generated RNAseq data can be compared? bioinforD Bioinformatics 6 11-25-2013 06:57 AM
Library prep issue nisha barak Sample Prep / Library Generation 2 11-19-2013 07:31 AM
looking for Solid library protocol casual_seqs SOLiD 12 05-09-2010 10:45 PM

Reply
 
Thread Tools
Old 07-07-2018, 08:21 AM   #1
eaakimov
Junior Member
 
Location: Helsinki

Join Date: Apr 2017
Posts: 1
Default RNASeq library generation protocol issue(like QuantSeq)

Hi,

I am trying to develop in-house protocol for RNA-seq library preparation in a way similar to Lexogen QuantSeq. QuantSeq includes double-stranded cDNA production with these major steps:
RT with dT(23)VN primer with overhang (i7 sequencing primer for NGS)
Removal of RNA (possibly with RNaseH)
Second strand synthesis with random primer with overhang (i5 seq primer for NGS)
These 3 steps are done without any purification. Then, they purify the product and perform PCR to add Illumina adapters.
I am wondering about step 3(second strand synthesis). I see the issue over here since dT(23)VN primer can also act as a primer for second strand synthesis. If so, during PCR to add Illumina adapters there could be an accumulation of a wrong product with two i7 adapters (adapters included in dT(23)VN primer). Any thoughts on how they overcome this issue? Thanks!
Short description for QuantSeq is here:
https://www.lexogen.com/wp-content/u...meth.f.376.pdf
eaakimov is offline   Reply With Quote
Old 07-07-2018, 04:22 PM   #2
nucacidhunter
Senior Member
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,166
Default

It should not be an issue as it requires polyA region for binding and priming. If there are such sequences (polyT) throughout the exons then some 1st strand cDNA might be primed but it will have negligible effect as these will not be sequenced due to lack of both flow cell binding motives.

Last edited by nucacidhunter; 07-07-2018 at 05:04 PM.
nucacidhunter is offline   Reply With Quote
Reply

Tags
ngs, rnaseq

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 10:00 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2018, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO